Vesicle-associated Membrane Protein 2 (VAMP2) but Not VAMP3 Mediates cAMP-stimulated Trafficking of the Renal Na+-K+-2Cl- Co-transporter NKCC2 in Thick Ascending Limbs

Background: Exocytic delivery of the renal co-transporter NKCC2 to the cell surface is a major mechanism for NaCl reabsorption. Results: We describe a mechanism that mediates cAMP-stimulated NKCC2 delivery in renal cells. Conclusion: Vesicle fusion protein VAMP2 interacts with NKCC2 and mediates cAMP-stimulated NKCC2 exocytic delivery. Significance: The molecular mechanism mediating NKCC2 exocytic delivery could provide new targets for treatment of hypertension. In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl(-) co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.